Study On Purification And Pharmacokinetics Of Phospholipid-Binding Anticoagulation Protein From Agkistrodon Halys Brevicaudus Venom

Objective 1. To purify Phospholipid-binding anticoagulation protein(PBAP) from Agkistrodon halys brevicaudus Venom by affinity chromatography and study its enzymatic characteristics. 2. To establish a double antibody sandwich enzyme-linked immunoadsorbent assay(ELISA) method for measuring the concentration of PhosphoLipid-binding anticoagulation protein(PBAP) from Agkistrodon halys brevicaudus Venom in rabbit serum. Compartment model was described and pharmacokinetic parameters were calculated according to the variation of serum concentrations,which provided experimental foundation for clinical research.Methods 1. By means of ion exchange chromatography and gel filtration,the PBAP was purified from Agkistrodon halys brevicaudus venom and used to immune the rabbits for preparing polyclonal antibody. The valence of the rabbit's antiserum was measured by ELISA .The titer and specificity of polyclonal antibody were detected by double immunodiffusion and western blotting .Anti-PBAP serum purified by precipitation method with ammonium sulfate was coupled to CNBr-activated Sepharose 4B. The PBAP was purified by Cation ion exchange chromatography on CM Sephadex C-25 ,affinity chromatography on Sepharose 4B and gel filtration on Sephacryl S-200.The enzymatic characteristics of purified PBAP were studied by the effects of PMSF,trypsin,aprotinin, bivalent metallic ion(Ca²⁺,Mg²⁺,Co²⁺,Mn²⁺,Zn²⁺),temperature,pH and EDTA on the AEHE activity of PBAP. 2. The purified PBAP from Agkistrodon halys brevicaudus venom was used to immune the rabbits for preparing antiserum.Anti-PBAP serum purified by precipitation method with ammonium sulfate was conjugated with horseradish peroxidase (HRP).The standard curve of double antibody sandwich ELISA was constructed ,and sensitivity ,accuracy and recovery of this method were calculated.PBAP was injected into auricular vein of rabbit with the dosage of 0.8 and 0.4mg·kg-1 respectively. 2ml of blood from common carotid artery was collected at every scheduled time(1,5,15,30,60,120,240 ,480min,respectively) after injection of PBAP,each serum was collected at 4℃.Serum concentrations were detected by double antibody sandwich ELISA method.Compartment model and pharmacokinetic parameters were calculated according to the variation of serum concentrations.Results 1. The PBAP and polyclonal antibody were successfully prepared after immunizing the rabbit.The valence of the antiserum and the titer of polyclonal antibody could achieve1︰12800,1︰32.The specificity of antibody was proved by western blotting. PBAP was isolated and purified from Agkistrodon halys brevicaudus venom by affinity chromatography which was successfully prepared by means of polyclonal antibody and CNBr-activated Sepharose 4B. PMSF and trypsin could inhibit the AEHE activity of PBAP. Aprotinin, bivalent metallic ion(Ca²⁺,Mg²⁺,Co²⁺,Mn²⁺,Zn²⁺)and EDTA had no effect on the AEHE activity of PBAP. The AEHE activity of PBAP was stable within 20～90℃and pH4.0～11.0 . 2. The PBAP of serum was specifically conjugated with polyclonal antibody.The standard curve of double antibody sandwich ELISA method showed that the minimal detection was 2.4ng·ml-1,and the curve exhibited linearity preferably within the range of 0.0024 to 10μg·ml-1.The average intra-assay and inter-assay coefficient of variance(CV) were 7.96% and 10.94%,the average recovery was 98.78%.The data of serum concentrations at different time points were analyzed with DAS2.0 ,the pharmacokinetics of PBAP at high and low doses were all consistented with two-compartment model.The main pharmacokinetic parameters of high-dose and low-dose groups were as follows:t1/2αwere 9.873±1.433min and 13.053±4.668min(P>0.05);t1/2βwere 205.798±76.834min and 241.295±66.06 min (P>0.05); AUC0-∞were 61.625±11.631 mg·L^-1·min and 27.114±2.781mg·L^-1·min(P<0.01); Cl were 0.014±0.003 L·min-1·kg-1 and 0.015±0.002 L·min-1·kg-1(P>0.05), respectively.Conclusion 1. The polyclonal antibody were successfully prepared after immuning the rabbit and affinity chromatography was a simple and rapid method which could purified PBAP from Agkistrodon halys brevicaudus Venom. 2. The double antibody sandwich ELISA showed preferably in accuracy,sensitivity and specificity.It was adopted in studying pharmacokinetics of PBAP in rabbits.